Kanamycin compound, kanamycin-producing Streptomyces species bacterium, and method of producing kanamycin

ABSTRACT

Vectors expressing kanA-kanB-kanK and other kanamycin production-related genes,  Streptomyces &lt;i/&gt; species recombinant bacteria transformed with the vectors, a method of producing kanamycin antibiotics by the bacteria, and a new kanamycin compound produced by the bacterium are provided. With the use of the recombinant bacteria of the present invention, the direct fermentative biosynthesis of amikacin and tobramycin as semi-synthetic kanamycins is possible, and the yield of kanamycin B as a precursor of the semi-synthetic kanamycin is improved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application filed under 35U.S.C. §371 of International Patent Application PCT/KR2010/007041,accorded an international filing date of Oct. 14, 2010; whichapplication claims priority to Korea Patent Application No.10-2010-0037709 filed Apr. 23, 2010 and Korea Patent Application No.10-2010-0082418 filed Aug. 25, 2010; which applications are incorporatedherein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 310179_(—)405USPC_SEQUENCE_LISTING.txt. The textfile is about 47 KB, created on Oct. 22, 2012, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to a new kanamycin compound, akanamycin-producing Streptomyces species bacterium, and a method ofproducing kanamycin using the bacterium.

BACKGROUND ART

Kanamycin produced by Streptomyces kanamyceticus is a 4,6-disubstituted2-deoxystreptamine-containing aminoglycoside antibiotic as aregentamicins and tobramycin, whereas neomycins and butirosins are4,5-disubstituted aminoglycosides.

These types of antibiotics are produced mainly by actinomycetes and havebeen widely used for a long time. Like other antibiotics, the appearanceof aminoglycoside-resistant bacteria, which produceaminoglycoside-modifying enzymes, cause serious problems. The issue ofresistance has been addressed by the semi-synthetic variants ofnaturally-occurring aminoglycosides such as amikacin, dibekacin, andarbekacin. The central 2-deoxystreptamine (2-DOS) andpseudodisaccharides 2′-N-acetylparomamine, paromamine, and neamine arecommon biosynthetic intermediates of most aminoglycosides. Thebiosynthetic route to neamine via 2-deoxystreptamine and paromamine waselucidated using recombinant enzymes involved in butiroin and neomycinbiosynthesis.

DISCLOSURE OF INVENTION Technical Problem

It has been reported a glycosyltransferase (GT) KanE (also called KanM2)from the kanamycin cluster, catalyzes the glycosylation of paromamine toproduce 3″-deamino-3″-hydroxykanamycin C using uridine5′-diphospho-D-glucose (UDP-Glc) as a cosubstrate. Alternatively, it isconsidered that kanamycin C and kanamycin B are produced as kanosamine(Kns: D-3-glucosamine) is transferred to paromamine and neamine,respectively, by KanE. Otherwise, it is considered that3″-deamino-3″-hydroxykanamycin C is a precursor of kanamycin C, whichcan produce kanamycin B as position C-6′ of kanamycin C is aminated. Itis also considered that kanamycin A can be synthesized by eitherKanE-catalyzed addition of kanosamine to 2′-deamino-2′-hydroxyneamine orby deamination of the position C-2′ of kanamycin B. However, thebiosynthesis of kanamycin from pseudodisaccharides or3″-deamino-3″-hydroxykanamycin C has not been well known. The reason forthis is that it is difficult to engineer aminoglycoside-producingactinomycetes genetically and obtain functional, soluble biosyntheticenzymes.

The inventors of the present invention have constructed an effectiveheterologous expression system based on an engineered strain of S.venezuelae and elucidated the biosynthesis of gentamicin A₂.

Therefore, an object of the present invention is to elucidate thebiosynthetic pathway of kanamycins and provide a method of producingkanamycin compounds and a new antibiotic produced by the same.

Solution to Problem

Recently, the inventors of the present invention isolated the kanamycinbiosynthetic gene cluster from S. kanamyceticus. The production ofkanamycin A by expression of this entire cluster in an aminoglycosidenon-producing S. venezuelae indicated it contains all of the genessufficient for the biosynthesis of the kanamycin complex.

Therefore, the object of the present invention can be achieved by avector comprising kanA-kanB-kanK and other kanamycin biosynthetic genes,a Streptomyces species bacterium transformed with the vector, a methodfor production of kanamycin by the bacterium, and a new kanamycincompound produced by the bacterium.

Advantageous Effects of Invention

According to the present invention, a kanamycin-producing Streptomycesspecies bacterium, a method for effective production of kanamycin, and anew kanamycin compound produced by the same are provided.

BRIEF DESCRIPTION OF DRAWINGS

The above and other features and advantages of the present inventionwill be more clearly understood from the following detailed descriptiontaken in conjunction with the accompanying drawing, in which:

FIG. 1A shows the structures of 2-DOS-containing aminoglycosides andFIG. 1B shows the previously proposed biosynthetic pathway ofkanamycins. In FIG. 1A, kanamycin, tobramycin, and gentamicin are4,6-disubstituted aminoglycosides, whereas butirosins and neomycins are4,5-disubstituted aminoglycosides. They are all naturally-occurringproducts, whereas amikacin, dibekacin, and arbekacin are semi-syntheticaminoglycosides derived from kanamycins. AHBA isS-4-amino-2-hydroxybutyric acid. FIG. 1B shows the biosynthesis ofcompound 5 (solid lines). However, a complete biosynthetic pathway(dotted lines) for compound 8 as a final product has not beendiscovered. 2-DOI: 2-deoxy-scyllo-inosose; 2-DOIA:2-deoxy-scyllo-inosamine; UDP-GlcNAc: uridine5′-diphospho-D-2-N-acetylglucosamine; and UDP-Glc: UDP-D-glucose.

FIGS. 2A to 2H show the results of HPLC-ESI-MS analysis of kanamycinbiosynthetic intermediates obtained in vitro expression of putativekanamycin gene sets in recombinant S. venezuelae hosts and in vitroreactions using cell-free extracts of recombinants. FIG. 2A shows thechromatograms of extracts from the recombinants (2-DOS, DOSf, and PAR)expressing kanA-kanB-kanK (2-DOS biosynthetic genes),kanA-kanB-kanK-kanF (kanF is a gene encoding the firstglycosyltransferase), and kanA-kanB-kanK-kanF-kacA (kacA is a geneencoding deacetylase). Each colored block represents an annotated gene.FIG. 2B shows the chromatograms of compounds 2 and 9 produced by thefirst glycosyltransferase KanF using three kinds of UDP sugars such asUDP-glucose (Glc), UDP-2-N-acetylglucosamine (GlcNAc), andUDP-2-glucosamine (GlcN) as substrates. Each box represents thecell-free extract obtained from the recombinant host expressing kanF,and black-framed boxes indicate the boiled cell-free extracts ascontrols. FIG. 2C shows the chromatograms of extracts from therecombinant (PARcd) expressing kanA-kanB-kanK-kanF-kacA and kanC-kanD atthe same time and the recombinant (PARil) expressingkanA-kanB-kanK-kanF-kacA and kanI-kacL at the same time. FIG. 2D showsthe chromatograms of compounds 4 and 10 produced by KanI-KacL-catalyzedreaction using paromamine and 2′-deamino-2′-hydroxyparomamine assubstrates. FIG. 2E shows the chromatograms of the extracts from therecombinant (KCXΔcΔd) expressing kanA-kanB-kanK-kanF-kacA-kanE, therecombinant (KCX) expressing kanA-kanB-kanK-kanF-kacA-kanE-kanC-kanD,the recombinant (KABΔcΔd) expressingkanA-kanB-kanK-kanF-kacA-kanE-kanI-kacL, and the recombinant (KAB)expressing kanA-kanB-kanK-kanF-kacA-kanE-kanC-kanD-kanI-kacL. FIG. 2Fshows the chromatograms of KanC-KanD and KanE-catalyzed production ofcompounds 5 and 6, supplemented with paromamine and UDP-Glc. Arrowsrepresent quenching of sequential reactions. FIG. 2G shows thechromatograms of KanC-KanD and KanE-catalyzed production of compounds 7and 13, supplemented with paromamine and UDP-Glc. FIG. 2H shows thechromatograms of the KanI-KacL-catalyzed production of compounds 7 and8.

FIG. 3 shows a decalcomania-like kanamycin biosynthetic pathway, inwhich each colored block represents an annotated gene and its producthas been proven to catalyze the biosynthesis of kanamycin intermediates.Each colored-circle represents the functional group formed by theproduct of the gene shown by the same colored block.

FIGS. 4A to 4C show the results of HPLC-ESI-MS/MS analysis of kanamycinbiosynthetic intermediates obtained from the recombinant strains inwhich the first glycosyltransferase-encoding gene has been changed. FIG.4A shows the chromatograms of extracts from the recombinants expressingkanA-kanB-kanK and kanF (DOSf), nemD (DOSn), tobM1 (DOSt₁) or gtmG(DOSg) at the same time, in which the bar graph on the right side showsthe yield of kanamycin pseudodisaccharide intermediates produced by eachrecombinant strain. The white bar indicates compound 2 and the gray barindicates compound 9. Data were obtained from triplicate analyses. FIG.4B shows the chromatograms of extracts from recombinants (KCX, KCXΔfn,KCXΔft₁, and KCXΔfg) expressing one ofkanA-kanB-kanK-kacA-kanE-kanC-kanD, together with kanF, nemD, tobM1, orgtmG. The bar graph shows the yield of kanamycin pseudodisaccharidesproduced by each recombinant strain. The white bar indicates compounds 5and 6, which are the congeners of compound 7 in the left-hand cycle ofthe decalcomania-like biosynthetic pathway. The gray bars indicateyields of compounds 11 and 12, which are the congeners of compound 8 inthe right-hand cycle. FIG. 4C shows the chromatograms of extracts fromrecombinants (KAB and KABΔfn) expressing one ofkanA-kanB-kanK-kacA-kanE-kanC-kanD-kanI-kacL together with kanE or nemD,in which the bar graph shows the yield of kanamycin produced by eachrecombinant. The white bars indicate the production of compounds 6 and7, and the gray bar represents the production of compounds 12 and 8.

FIGS. 5A to 5C show the results of HPLC-ESI-MS/MS analysis of compounds17, 19, and 20 produced by the recombinant strains. FIG. 5A shows thechromatograms of the products from the recombinant (KCXb) expressing acompound 12-producing gene set (kanA-kanB-kanK-kacA-kanE-kanC-kanD)together with seven butirosin biosynthetic genes(btrG-btrH-btrI-btrJ-btrK-btrO-btrV) or the recombinant (KABb)expressing a compound 8-producing gene set(kanA-kanB-kanK-kacA-kanE-kanC-kanD-kanI-kacL). Compound 15 is theAHBA-conjugated kanamycin derivative amikacin and compound 17 is thepreviously uncharacterized AHBA-conjugated kanamycin derivative1-N-AHBA-kanamycin X. FIG. 5B shows the chromatograms of the productsfrom the recombinants (KABΔfna and KABΔfnΔet₂a) expressing aprD3-aprD4and a compound 8-biosynthetic gene set such askanA-kanB-kanK-nemD-kacA-kanE-kanC-kanD-kanI-kacL orkanA-kanB-kanK-nemD-kacA-tobM2-kanC-kanD-kanI-kacL at the same time.Compounds 16 and 18 are tobramycin and nebramine as a tobramycinbiosynthetic intermediate. FIG. 5C shows the structures of kanamycinanalogs produced by the recombinants expressing aprD3-aprD4 and tobM2genes. Each colored-circle represents the functional group formed by theproduct of the gene with annotation information shown by the samecolored block.

FIGS. 6A and 6B show the results of HPLC-ESI-MS/MS analysis of kanamycinpseudodisaccharides obtained from in vitro reactions using cell-freeextracts of recombinant S. venezuelae strains expressing kanE andkanC-kanD, respectively. FIG. 6A shows the chromatograms of theproduction of compounds 11 and 12. Each box represents the cell-freeextract obtained from the recombinant host expressing kanE or kanC-kanD,black-framed boxes indicate the boiled cell-free extracts as controls,and arrows represent quenching of sequential reactions. FIG. 6B showsthe chromatograms of the production of compounds 8 and 14.

FIG. 7 shows the results of HPLC-ESI-MS/MS analysis of compounds 16 and18 produced from in vitro reactions using cell-free extracts ofrecombinant S. venezuelae strains expressing aprD3-aprD4 gene pair. Eachbox represents the cell-free extract obtained from the recombinant hostexpressing aprD3-aprD4 gene pair, and black-framed boxes indicate boiledcell-free extracts as controls.

FIG. 8 shows the results of HPLC-ESI-MS/MS analysis of compounds 16produced from in vitro reactions using cell-free extracts of wildstrains of S. kanamyceticus and Streptomyces tenebrarius. Each boxrepresents the cell-free extract obtained from S. kanamyceticus ATCC12853 (SK CFE) and S. tenebrarius ATCC 17920 (ST CFE), and black-framedboxes indicate boiled cell-free extracts as controls.

FIG. 9 shows the results of HPLC-ESI-MS/MS analysis of kanamycinpseudodisaccharides obtained from in vitro reactions using cell-freeextracts of wild-type S. kanamyceticus strain. Each box represents thecell-free extract obtained from the S. kanamyceticus ATCC 12853 (SKCFE), and black-framed boxes indicate boiled cell-free extracts ascontrols.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a vector comprising at least one gene setselected from the group consisting of:

(1) kanA, kanB, and kanK (DOS strain);

(2) kanA, kanB, kanK, and kanF (DOSf strain);

(3) kanA, kanB, kanK, kanF, and kacA (PAR strain);

(4) kanA, kanB, kanK, kanF, kacA, kanC, and kanD (PARcd strain);

(5) kanA, kanB, kanK, kanF, kacA, kanI, and kacL (PARil strain);

(6) kanA, kanB, kanK, kanF, kacA, and kanE (KCXΔcΔd strain);

(7) kanA, kanB, kanK, kanF, kacA, kanE, kanC, and kanD (KCX strain);

(8) kanA, kanB, kanK, kanF, kacA, kanE, kanI, and kacL (KABΔcΔd strain);

(9) kanA, kanB, kanK, kanF, kacA, kanE, kanC, kanD, kanI, and kacL (KABstrain);

(10) kanA, kanB, kanK, kanF, kacA, kanE, kanC, kanD, btrG, btrH, btrI,btrJ, btrK, btrO, and btrV (KCXb strain);

(11) kanA, kanB, kanK, kanF, kacA, kanE, kanC, kanD, kanI, kacL, btrG,btrH, btrI, btrJ, btrK, btrO, and btrV (KABb strain);

(12) kanA, kanB, kanK, and nemD (DOSn strain);

(13) kanA, kanB, kanK, nemD, kacA, kanE, kanC, and kanD (KCXΔfn strain);

(14) kanA, kanB, kanK, nemD, kacA, kanE, kanC, kanD, kanI, and kacL(KABΔfn strain);

(15) kanA, kanB, kanK, nemD, kacA, kanE, kanC, kanD, kanI, kacL, aprD3,and aprD4 (KABΔfna strain);

(16) kanA, kanB, kanK, nemD, kacA, tobM2, kanC, kanD, kanI, kacL, aprD3,and aprD4 (KABΔfnΔet₂a strain);

(17) kanA, kanB, kanK, and tobM1 (DOSt₁ strain);

(18) kanA, kanB, kanK, tobM1, kacA, kanE, kanC, and kanD (KCXΔft₁strain);

(19) kanA, kanB, kanK, and gtmG (DOSg strain); and

(20) kanA, kanB, kanK, gtmG, kacA, kanE, kanC, and kanD (KCXΔfg strain),and

a kanamycin-producing recombinant Streptomyces species bacteriumtransformed with the vector.

Preferably, the kanA, kanB, kanK, kanF, kacA, kanE, kanC, kanD, kanI,and kacL are derived from Streptomyces kanamyceticus and have sequencenumbers 5 to 14.

The aprD3, aprD4, tobM1, and tobM2 are derived from Streptomycestenebrarius and have sequence numbers 15 to 18.

The btrG, btrH, btrI, btrJ, btrK, btrO, and btrV are derived fromBacillus circulans and have sequence numbers 19 to 25.

The nemD is derived from Streptomyces fradiae and has sequence number26.

The gtmG is derived from Micromonospora echinospora and has sequencenumber 4.

Preferably, the Streptomyces species bacterium further expresses akanamycin-resistant gene. Preferably, the kanamycin-resistant genecomprises gtmF, gtmK, and gtmL derived from Micromonospora echinospora,which have sequence numbers 1 to 3.

The Streptomyces species bacterium is Streptomyces venezuelae, but notlimited thereto. Preferably, the Streptomyces species bacterium isStreptomyces venezuelae KCTC11725BP and, more preferably, theStreptomyces species bacterium is Streptomyces venezuelae KCTC11726BP.The Streptomyces species bacterium is Streptomyces venezuelaeKCTC11727BP and, preferably, the Streptomyces species bacterium isStreptomyces venezuelae KCTC11728BP.

Moreover, the present invention provides a method of producing akanamycin antibiotic using the Streptomyces species bacterium, and thekanamycin antibiotic produced by the present invention comprises thefollowing compounds 1 to 20:

Compound 1: 2-deoxystreptamine (2-DOS)

Compound 2: 2′-N-acetylparomamine;

Compound 3: paromamine;

Compound 4: neamine;

Compound 5: 3″-deamino-3″-hydroxykanamycin C;

Compound 6: kanamycin C;

Compound 7: kanamycin B;

Compound 8: kanamycin A;

Compound 9: 2′-deamino-2′-hydroxyparomamine;

Compound 10: 2′-deamino-2′-hydroxyneamine

Compound 11: 3″-deamino-3″-hydroxykanamycin X;

Compound 12: kanamycin X;

Compound 13: 3″-deamino-3″-hydroxykanamycin B;

Compound 14: kanamycin D;

Compound 15: amikacin;

Compound 16: tobramycin;

Compound 17: 1-N-AHBA-kanamycin X;

Compound 18: nebramine;

Compound 19: 3′-deoxykanamycin C; and

Compound 20: 3′-deoxykanamycin A.

Moreover, the products obtained from the recombinant bacteria are asfollows:

DOS: compound 1;

DOSf: compounds 2 and 9;

PAR: compounds 3 and 9;

PARcd: compounds 3 and 9;

PARil: compounds 4 and 10;

KCXΔcΔd: compounds 5 and 11;

KCX: compounds 6 and 12;

KABΔcΔd: compounds 13 and 14;

KAB: compounds 7 and 8;

KCXb: compound 17;

KABb: compound 15;

DOSn: compounds 2 and 9;

KCXΔfn: compounds 6 and 12;

KABΔfn: compounds 7 and 8;

KABΔfna: compounds 18, 19 and 20;

KABΔfnΔet₂a: compound 16;

DOSt₁: compounds 2 and 9;

KCXΔft₁: compounds 6 and 12;

DOSg: compounds 2 and 9; and

KCXΔfg: compounds 6 and 12.

When the kanamycin is produced by the gene recombinant bacteria of thepresent invention, especially KABΔfn strain, the yield of kanamycin B asa precursor of arbekacin, which is one of the semi-syntheticaminoglycosides, is improved compared to wild-type strains.

Moreover, with the use of the gene recombinant bacteria of the presentinvention, especially KABb and KABΔfnΔet₂a strains, it is possible toproduce amikacin and tobramycin by direct fermentation, not bysemi-fermentation and semi-synthesis.

Furthermore, the present invention provides a new compound representedby the following formula 1:

The new compound is compound 17, 1-N-AHBA-kanamycin X.

The kanamycin compound of the present invention produced by thebacterium of the present invention has an antibacterial effect. Moreparticularly, the compound of the present invention has an antibacterialeffect on Gram-negative bacteria. The compound has an antibacterialeffect on Pseudomonas aeruginosa and Escherichia coli, but not limitedthereto. Especially, the new compound, 1-N-AHBA-kanamycin X, has anantibacterial effect on amikacin-resistant P. aeruginosa.

Recently, various studies have reported that aminoglycoside antibioticscontaining the 2-DOS have antiviral effects on various viruses such asbovine viral diarrhea virus (BVDV), dengue virus (DENY), etc. Moreover,many studies have reported that the aminoglycoside antibiotics, whichinduce mammalian ribosomes to read-through premature stop codonmutations, can treat and improve hereditary diseases such as cysticfibrosis, muscular atrophy, Hurler syndrome, telangiectasis, and Ushersyndrome. The kanamycin compound of the present invention also containsthe 2-DOS as a fundamental structure, and thus it is considered that thekanamycin compound of the present invention has an antiviral effect andcan treat and improve such hereditary diseases.

Therefore, the present invention provides an antibacterial compositioncomprising the kanamycin compound as an effective ingredient.

Moreover, the present invention provides an antiviral compositioncomprising the kanamycin compound as an effective ingredient.

Furthermore, the present invention provides a pharmaceutical compositioncomprising the kanamycin compound as an effective ingredient for thetreatment of at least one disease selected from the group consisting ofcystic fibrosis, muscular atrophy, Hurler syndrome, telangiectasis, andUsher syndrome.

The composition may be administered orally, rectally, vaginally,topically, transdermally, intravenously, intramuscularly,intraperitoneally, or subcutaneously. The effective dose of the activecompound may vary depending on the minimum inhibitory concentration(MIC), the age and condition of the patient, particular disease, theseverity of disease or condition, the route of administration, andadministrator s decision.

The pharmaceutical composition may be medicated in the form of solid,semi-solid, or liquid depending on an administration pattern. It mayinclude tablets, powders, suppositories, granules, creams, lotions,ointments, patches, aqueous solutions, suspensions, dispersions,emulsions, syrups, but are not limited to them. Active ingredients canbe encapsulated in liposomes, nanoparticles, microcapsules, etc. Solidcompositions such as tablets, pills, granules, etc. may be coated forconvenience.

The composition of the present invention may contain pharmaceuticallyacceptable excipients such as diluents, carriers, isotoners,stabilizers, antioxidants, binders, colorants, flavorings,preservatives, thickening agents, etc. Such excipients can be selectedby one skilled in the art according to the administration routes andformulations. Moreover, the composition of the present invention maycontain a small amount of a non-toxic adjuvant such as a wetting agent,emulsifier, pH buffer agent, etc. The non-toxic adjuvant may include,but not limited to, acetate, sorbitan monolaurate, triethanolamine, andtriethanolamine oleate. The composition for intravenous administrationmay be a solution in sterile isotonic aqueous buffer solution and maycontain a local anesthetic for relief of pain in the injection area.

TABLE 1 Deoxyoligonucleotide primers used for PCR amplification of genesPrimer sequence(5′ to 3′, Portion restriction site shown of Enzyme Genewith underline) gene site kanA-kanB cccggatccgaatcccccttcgtgacg 5′ BamHIgtgactagtgttcgtcgaccaccgcgtcga 3′ SpeI kanK gcttctagaactccggagcacccgtgca5′ XbaI gtgaagcttcgtggtgccggacaggcccta 3′ HindIII kanCgggacctctagaacgcggtggtcgacgaac 5′ XbaItcttccaagcttactagttgtcggcggtcgccccga 3′ SpeI/HindIII kanDgaccgctctagacacctccgaggtcctctc 5′ XbaItgaaaagcttactagtgggtgacgagacgccggg 3′ SpeI/HindIII kanEacatctagaggctccggaagaccgccgacgcca 5′ XbaItcgaagcttactagtgagacgaggaggaccctt 3′ SpeI/HindIII kanFcgaggctctagagccggaccagaacccatt 5′ XbaItagaacaagcttactagtacgtcgggtgtcgtacgg 3′ SpeI/HindIII kanI-kacLgatcgctctagacacctggttctggttccc 5′ XbaIaggggaaagcttactagtgtcaggagatgccgaccg 3′ SpeI/HindIII kacAggcgtctagataccgggagatcgggctgtg 5′ XbaIacaaagcttactagttgctcatagcgactccttgt 3′ SpeI/HindIII nemDcactctagacatcgccgtcctctccccgt 5′ XbaI tcaaagcttactagtggcgcagatacggcgcac3′ SpeI/HindIII tobM1 ccgtctagaccgccctttccccgcaac 5′ XbaIgataagcttactagtcacaccgtcctattcctg 3′ SpeI/HindIII tobM2cgatctagaccgggaggcgaccgtgt 5′ XbaI gcgaagcttactagttcagacgcatacgcccag 3′SpeI/HindIII gtmG tgtcctctagagctgcccggtcacttcccgc 5′ XbaIaaaaagcttactagtcactcttccggaagaatc 3′ SpeI/HindIII btrG-btrHttctctagatctaggaaaccgcatgcc 5′ XbaI ggctacgtaaaactagtggtttatccgcttttgct3′ SpeI/SnaBI btrI-btrJ acctctagacggaaaccgccatcccat 5′ XbaItcctacgtaaaactagtgagttaatgaacagccgt 3′ SpeI/SnaBI btrK-btrOagctctagaaagcccgaagcctgcttg 5′ XbaI gtttacgtaaaactagttccctagaccggattcga3′ SpeI/SnaBI btrV tattctagaattgacttataactcaat 5′ XbaIcattacgtaaaactagtatccgaacgtcacataag 3′ SpeI/SnaBI aprD3ggttctagatgatggacgtggccgcga 5′ XbaI cttaagcttactagtgggccgtcggtcgtcctg 3′SpeI/HindIII aprD4 ggctctagaaaccccaccctcaccatccag 5′ XbaIcgcaagcttactagtatcgtgtggtctccggct 3′ SpeI/HindIII

The inventors of the present invention have reconstructed a completebiosynthesis of kanamycin by integrating the plasmid containing variouscandidate genes isolated from S. kanamyceticus into S. venezuelaestrain, which is deficient in biosynthesis of the endogenous deoxysugarthymidine 5′-diphospho (TDP)-D-desosamine. The inventors have amplifiedputative kanamycin biosynthetic genes by PCR with specific primers shownin table 1.

The inventors have modified the kanamycin biosynthetic flux to producekanamycin B (compound 7) as a main product. The reason for this is thatthe production of compound 7 leads to the production of3′-deoxykanamycin and 1-N-AHBA kanamycin by direct fermentation, whichwill be valuable substitutes for the chemical synthesis of dibekacin andarbekacin.

After examining the kanamycin analogs obtained from the recombinants andtheir structures, it has been confirmed that the kanamycin antibiotic,which is known as one of the compounds produced from the recombinantbacteria of the present invention, has the same properties as thecommercially available standard kanamycin analog or the kanamycin analogderived from S. kanamyceticus. However, it has been ascertained that1-N-AHBA-kanamycin X, a new kanamycin analog, has been produced by therecombinant heterologous strain of the present invention in addition tothe conventional kanamycin antibiotics.

First, compound 3 was biosynthesized from Glc-6-phosphate as shown inFIG. 1B. As expected, the recombinant heterologous strain expressingkanA-kanB-kanK (DOS strain) produced compound 1 (21.4 μM) (FIG. 2A). Toconfer resistance to kanamycin in S. venezuelae without modifying theaminoglycoside structure, three resistance genes gtmF-gtmK-gtmL from thegentamicin gene cluster were introduced into the recombinantheterologous strain. Transformants expressing these genes were resistantto >10.0 mg/ml of the commercially available kanamycin compared to 1.0μg/ml in control strains. These resistance genes were expressed togetherwith the kanamycin biosynthetic genes in the heterologous host insubsequent experiments.

Compound 2 (1.3 μM) was produced by expressing DOS biosynthetic genesplus kanF encoding the first glycosyltransferase. Interestingly, agreater amount of compound 9 (7.1 μM) was produced at the same time.Moreover, The cell-free extract of S. venezuelae expressing kanFsupplemented with compound 1 as a glycosyl acceptor and UDP-Glc orUDP-GlcNAc as a glycosyl donor produced 9-fold greater amount ofcompound 9 than that of compound 2 (FIG. 2B). Therefore, it indicatesthat KanF accepts both UDP-Glc and UDP-GlcNAc as cosubstrates butpreferentially transfers UDP-Glc to compound 1. Compound 3 (2.2 μM) wasproduced by the recombinant strain (PAR strain) expressing kacA as2′-N-acetylparomamine deacetylase together with compound 2/9biosynthetic genes (FIG. 2A).

It is necessary to convert compound 3 into compound 4 for the kanamycinbiosynthesis. It was estimated that KanI and KacL would participate inthe conversion of compound 3 into compound 4 based on the in silicoanalysis of the aminoglycoside gene cluster. Meanwhile, it is consideredthat KanC and KanD are responsible for the formation of UDP-kns byaminating the C-3 hydroxyl group of UDP-Glc. Otherwise it is consideredthat KanC and KanD participate in the production of compound 6 fromcompound 5.

In one example of the present invention, kanC-kanD genes were firstexpressed in PAR strain (PARcd strain) to identify the functions ofKanI-KacL and KanC-KanD. However, expression of kanC-kanD in the strainPAR (strain PARcd) produced only 3 and 9 (FIG. 2C), indicating thatKanC-KanD does not participate in amination of the pseudodisaccharides.On the contrary, the recombinant strain (PARil strain) expressingkanI-kacL produced compound 10 (4.3 μM) as well as compound 4 (2.2 μM)(FIG. 2C). Incubation of the cell-free extracts of the recombinantexpressing kanI-kacL with compounds 3 and 9 resulted in the productionof compounds 4 and 10 (FIG. 2D).

In one example of the present invention, pseudotrisaccharide kanamycinswere biosynthesized. Compound 5 (3.2 μM) and compound 11 (4.5 μM), whichwas not known as a biosynthetic intermediate, were produced by therecombinant (KCXΔcΔd strain) expressing kanE encoding a secondglycosyltransferase together with genes for biosynthesis of compound 3/9(FIG. 2E). Additional expression of kanC-kanD in KCXΔcΔd strain (KCXstrain) resulted in the production of compound 6 (1.6 μM) and compound12 (6.0 M), C-3″ amination. The strain (KABΔcΔd strain) obtained byexpressing kanI-kacL in KABΔcΔd strain produced compound 13 (2.0 μM) andcompound 14 (3.2 μM). Moreover, the strain (KAB strain) obtained byexpressing kanC-kanD in KABΔcΔd strain produced compound 7 of 1.0 mg/l(2.1 μM) and compound 8 of 3.1 mg/l (6.3 μM) (FIG. 2E).

In one example of the present invention, cell-free extracts ofrecombinant S. venezuelae strains expressing kanE or kanC-kanD wereprepared to determine whether UDP-kns biosynthesized from UDP-Glc islinked at the C-6 position of pseudodisaccharide or whether a Glc moietyof pseudodisaccharide is converted into kanosamine. Compounds 3 and 4were incubated with UDP-Glc to produce compounds 5/6 and 13/7,respectively. When compounds 3 and 4 and UDP-Glc were added to thecell-free extracts of the recombinant strain expressing kanE, compounds5 and 13 were produced, respectively. When these reactions were quenchedand the resulting mixture was incubated together with the cell-freeextracts of the strain expressing kanC-kanD, compounds 5 and 13 remainedand further conversion was not observed. However, when compounds 3 and 4were reacted with UDP-Glc and the cell-free extracts of the strainexpressing kanC-kanD, compounds 6 and 7 were produced, respectively(FIGS. 2F and 2G). The same results were observed when compounds 9 and10 were used as substrates (FIG. 6). These results showed that thehydroxyl group of UDP-Glc was aminated before the glycosyl transferreaction by the enzyme pair during the biosynthesis of kanosamine. Thisexample can be observed in the biosynthesis of kanosamine inAmycolatopsis mediterranei, a rifamycin-producing strain.

Moreover, when the cell-free extracts of the strain expressing kanI-kacLwere incubated together with compounds 6 and 12, compounds 7 and 8 wereproduced, respectively. It is considered that KanI and KacL participatein the biosynthesis of compounds 4 and 10 as well as compounds 7 and 8(FIGS. 2D and 2H), which shows their substrate flexibility towardpseudodi- and tri-saccharides. In summary, a decalcomania-like kanamycinbiosynthetic pathway of the kanamycin complex dominated by KanF and KanEglycosyltransferases was identified (FIG. 3).

In one example of the present invention, the biosynthesis of kanamycinwas modified. Compound 8 is the major fermentation product in bothwild-type kanamycin producer S. kanamyceticus and the heterologous hostS. venezuelae. It was shown that KanF glycosyltransferase preferredUDP-Glc to UDP-GlcNAc in the previous experiment. KanI-KacL, whichpreferred compound 12 to compound 6, was involved in the yield ofcompounds 8 and 7 (FIG. 2H). While amikacin (compound 15) can besynthesized from compound 8, the arbekacin, a latest semi-syntheticaminoglycoside having antibacterial activity against resistant bacteria,can be produced by removing the 3′,4′-hydroxyl group followed by1-N-acylation with AHBA. It was supposed that the biosynthesis ofkanamycin tends to increase the yield of compound 7, if the kanF geneencoding the first glycosyltransferase was substituted with another geneencoding the glycosyltransferase which prefers UDP-GlcNAc to UDP-Glc asa glycosyl donor in the genetic construct for the biosynthesis ofcompounds 7 and 8. First, the inventors replaced kanF in the DOS strainwith nemD, tobM1, and gtmG encoding three different glycosyltransferasesfrom neomycin, tobramycin, and gentamicin. The DOSf strain as a controlexpressing kanF produced compound 2 (1.4 μM; 14% of the totalpseudodisaccharide) and compound 9 (7.1 μM; 79%). On the contrary, thestrain (DOSn strain) expressing nemD produced compound 2 (4.4 μM; 57%)and compound 9 (3.4 μM; 36%), from which it seemed that NemD useUDP-GlcNAc preferentially in contrast to KanF. When tobM1 and gtmGreplaced kanF (DOSt₁ and DOSg strains), there was no significant changein the ratio of compounds 2 and 9 produced (FIG. 4A).

Moreover, in another example of the present invention, nemD, tobM1, andgtmG were separately substituted for kanF in KCX strain. When comparingthe ratio of compounds 6 and 12 (1.6 μM, 16% and 6.0 μM, 60%) producedby KCX strain, the ratio of pseudodisaccharides 6 and 12 (5.0 μM, 65%and 0.4 μM, 7%) produced by the recombinant strain (KCXΔfn) expressingnemD was inverted. However, the ratio of pseudodisaccharide products 6and 12 in the recombinant strain expressing either tobM1 (KCXΔft₁) orgtmG (KCXΔfg) were similar to each other (FIG. 4B). Moreover, while theyields of compounds 7 and 8 produced by KAB strain were 21% (2.3 μM) and61% (6.8 μM), the yield of compound 7 in the strain (KABΔfn) obtained bysubstituting kanF with nemD was increased to 46% (FIG. 4C).

In another example of the present invention, 1-N-AHBA-kanamycin and3′-deoxykanamycin were produced by direct fermentation. The inventorshave modified the kanamycin biosynthetic pathway for in vivo productionof 1-N-acylated kanamycin containing AHBA such as compound 15 and3′-deoxykanamycin such as compound 16. The AHBA molecular structure wasobserved in the naturally-occurring butirosin. Recently, it has beenreported that the btrG-btrH-btrI-btrJ-btrK-btrO-btrV gene set isresponsible for biosynthesis and introduction of the AHBA side chaininto the amino group at C-1 of 2-DOS in butirosin. Compound 17 (0.6mg/l, 1.0 μM), a new aminoglycoside compound, was produced byintroducing these genes into the KCX strain. Moreover, 0.5 mg/l (0.8 μM)of compound 15 was successfully produced by expressing the btr gene setin the KAB strain (FIGS. 5A and 5C).

Moreover, the inventors have analyzed the aminoglycoside gene cluster bybioinformatics analysis to find aprD3-aprD4, two putative apramycingenes, from S. tenebrarius, a tobramycin (compound 16)-producing strain.It seems that the aprD3-aprD4 genes are responsible for C-3′deoxygenation of pseudodi- and/or tri-saccharides.

In one example of the present invention, the aprD3-aprD4 genes wereintroduced into KABΔfn engineered for the increased production ofcompound 7. The resulting strain KABΔfna produced compound 18 as a majorproduct and produced compounds 19 and 20 but did not produce compound 16(FIGS. 5B and 5C). This result shows that the activity of AprD3-AprD4pair removes the 3′-hydroxyl group in compounds 3, 10, and 4. It alsoindicates the second glycosyltransferase KanE selectively transferskanosamine to compounds 3 and 10 but not to compound 18. To select theglycosyltransferase that transfers kanosamine to compound 18 to producecompound 16, cell-free extracts of S. kanamyceticus and S. tenebrariuswere prepared and incubated together with compound 18. The cell-freeextract of S. tenebrarius containing tobM2 as a secondglycosyltransferase encoding gene converted compound 18 into compound16, but the cell-free extract of S. kanamyceticus did not (FIG. 7).Therefore, the inventor have constructed the glycosyltransferaseencoding genes nemD, tobM2, and aprD3-aprD4 and a new strain(KABΔfnΔet₂a) expressing compound 7 biosynthetic gene and producedcompound 16 (0.4 mg/1, 0.8 μM) (FIGS. 5B and 5C). The cell-free extractsof the strain expressing aprD3-aprD4 converted compound 4 into compound18, but did not convert compound 7 into compound 16, which means thatthese enzymes are active only to pseudodisaccharides (FIG. 7).

In one embodiment of the present invention, antibacterial spectraagainst kanamycin intermediates and analogs produced by the presentinvention were measured. Typical gram-negative strains (E. coli andPseudomonas aeruginosa) and four clinically isolated strains wereemployed to check the antibacterial spectra (See table 2).

Among the pseudodisaccharides, the 6′-amino compounds 4 and 10 were moreactive than the 6′-hydroxy derivatives 3 and 9 againstkanamycin-sensitive E. coli strains. Compared with compounds 6 and 12containing 6′-hydroxy group, their 6′-amino counterparts 7 and 8 weremore active against kanamycin-sensitive test strains. In addition, the3″-amino compounds 6, 7, 8, and 12 showed increased activity against thetest strains when compared with the corresponding 3″-hydroxy derivatives5, 13, 14, and 11. Therefore, when C-6′ and/or C-3″ hydroxyl group ofkanamycin were aminated, the antibacterial activity was increased, whichwas consistent with the previous study.

In the test with the clinically isolated amikacin-sensitive P.aeruginosa strain, most kanamycin intermediates showed very lowantibacterial with exception of the compounds 6, 7, 8, and 12.Interestingly, new compound 17 showed improved antibacterial activityagainst all the test strains compared with amikacin (compound 15).Especially, compound 15 showed no activity on the clinically isolatedamikacin-resistant P. aeruginosa strain, whereas, compound 17 showedvery strong activity (MIC ˜64).

TABLE 2 Antibacterial spectra against Gram-negative bacteria MIC (μg/ml)Typical strains Clinically isolated strains E. coli P. aeruginosa E.coli E. coli P. aeruginosa P. aeruginosa Kanamycin-related ATCC ATCCCCARM CCARM CCARM CCARM aminoglycosides 25922(Kan^(S)) 27853(Kan^(R))1A020(Kan^(S)) 1A023(Kan^(R)) 2206(Amk^(S)) 2178(Amk^(R)) Pseudodi- 3128 NA 128 NA NA NA saccharides 9 128 NA 128 NA NA NA 4 64 NA 64 NA 128NA 10 64 NA 64 NA 128 NA Pseudotri- 6 16 NA 16 NA 64 NA saccharides 1216 NA 16 NA 64 NA 5 128 NA NA NA NA NA 11 NA NA NA NA NA NA 7 2 NA 4 NA32 NA 8 2 NA 4 NA 16 NA 13 128 NA 128 NA NA NA 14 128 NA 128 NA NA NAAHBA- 15 128 64 128 32 ≦0.25 NA pseudotri- 17 16 16 32 16 0.25 64saccharides MIC: minimum inhibitory concentration. Type strains andclinically isolated strains were obtained from ATCC (American TypeCulture Collection, USA) and CCARM (Culture Collection of AntimicrobialResistant Microbes, Republic of Korea), respectively. Kan^(S) andKan^(R) represent the kanamycin-sensitive strain and thekanamycin-resistant strain, respectively. Amk^(S) and Amk^(R) representthe amikacin-sensitive strain and the amikacin-resistant strain,respectively. NA represents no activity at 128 μg/mL.

The inventors have constructed 3D models of glycosyltransferases such asKanF and NemD. The 3D models were generated by homology modeling usingthe crystal structure of the MshA as a template. These enzymes sharedover 40% overall sequence similarity, respectively. Critical residuesand their interactions were preserved in both strongly supportingconserved catalytic association mechanisms.

To gain insight into the catalytic mechanism, the simulations aimed atthe preparation of KanF/NemD complex with glycosyl-donors/acceptor wererepeated. First, the inventors performed a docking of theglycosyl-donors (UDP-Glc and UDP-GlcNAc) to the putative substratebinding site in both glycosyltransferases KanF and NemD using AutoDock,respectively. Secondly, the glycosyl-acceptor (compound 1) was dockedinto the putative glycosyl-acceptor binding site of eachglycosyltransferase/glycosyl-donors complex using CDOCKER and deeplydocked into the Glc or GlcNAc area, thus making ideal interactions withtheir glycosyl-donors and the surrounding residues of eachglycosyltransferase.

The distance between the C1 atom of the glycosyl-donors and O3 atom ofcompound 1 fluctuated only within very narrow limits (˜3.0 Å). The totalenergy of the [KanF or NemD/glycosyl-donors/compound 1] complex wasstabilized and remained stable during the simulation. The radius ofgyration for the complexes was found to be stable at roughly 20 Åthroughout the simulation, suggesting that the presence of compound 1 inthe complexes induce the protein to adopt more compact form compared tothe proteins in the absence of compound 1. There is a large hydrophilicpocket between two domains of glycosyltransferase KanF (or NemD). Thepocket is formed by glycosyl-donor neighboring hydrophilic residues suchas Glu14 (Glu41), Gln19 (Gln46), Gln20 (Gln47), His88 (His115), Tyr110(Tyr137), Thr111 (Thr138), Asp117 (Asp204), Lys218 (Lys245), Asn272(Asn299), Glu292 (Glu319), Glu293 (Glu320), Ser297 (Ser324), and Glu300(Glu327). Analysis of hydrophilic-hydrophilic contact in the dockedstructure showed that these residues are playing a crucial role in theinteraction. The sugar groups of glycosyl-donors/compound 1 wouldinteract with hydrophilic residues of the binding pocket. The uracilmoiety of both glycosyl-donors forms hydrophobic interactions withGly243 (Gly270), Phe269 (Phe296), and Ile275 (Ile302), whereas theirribose moieties build the hydrophilic interactions with Ser297 (Ser324),and Glu300 (Glu327). The hydrophilic contact between compound 1 andTyr110 (Tyr137) is strong because the phenolic group of Tyr is movedsomewhat inward or outward from its original position as compound 1which is located above the phenolic group tries to get closer to Tyr110(Tyr137). However, Leu89 (Leu116) make a strong hydrophobic interactionwith compound 1 which doesn't move much at all. The amino group at C3position in compound 1 interacts with the Asp177 (Asp204) which allowsthe dissipation of the delocalized negative charge of compound 1. Thehydroxyl group at C6 position in compound 1 make hydrophilicinteractions with His88 (His115), whereas the ring moiety of compound 1makes hydrophobic interactions with Gly16 (Gly43) and Val17 (Val44). TheGlc or GlcNAc part in the glycosyl-donor is anchored to a pocket formedby Glu292 (Glu319), Glu293 (Glu320), and Leu294 (Leu321), proposing thatthese residues might play a critical role in the stability of thosecomplexes. The sugar parts of glycosyl-donors stack against between thecarboxyl moieties of Glu292 (Glu319) and Glu293 (Glu320). Leu294(Leu321) is located at almost the center of hydrophobic and hydrophilicinteraction with sugar moiety.

The inventors have performed the molecular dynamics and binding freeenergy analysis. The glycosyl-donors have several hydrophilic groups asdescribed above, thus their hydrogen bond interaction withglycosyltransferases as well as solvent molecules at the active site isimportant for them to bind tightly with both KanF and NemD. The numberof hydrogen bonds between KanF and UDP-Glc (˜5) is larger than thatbetween KanF and UDP-GlcNAc (˜2), whereas the number between NemD andUDP-Glc (˜3) is smaller than that between NemD and UDP-GlcNAc (˜6),indicating that UDP-Glc binds more tightly to KanF than does UDP-GlcNActo NemD.

The calculated free energies of UDP-Glc and UDP-GlcNAc binding to KanFand NemD is −42.2, −47.3, −41.4, and −46.7 kJ/mol, respectively (Seetable 3).

TABLE 3 Average binding free energies(kJ/mol) between glycosyl-donorsand surrounding residues of KanF and NemD during molecular dynamicssimulation. KanF + KanF + NemD + NemD + UDP-GlcNAc UDP-Glc UDP-GlcUDP-GlcNAc $\left\langle {E\frac{vdW}{enz}} \right\rangle$  −7.98 ± 0.59 −7.13 ± 0.17  −8.07 ± 0.24  −7.45 ± 0.40$\left\langle {E\frac{vdW}{{enz} + {sub}}} \right\rangle$  −42.09 ± 0.39 −35.04 ± 0.84  −43.90 ± 0.38  −41.95 ± 0.08$\left\langle {E\frac{Elec}{enz}} \right\rangle$ −258.33 ± 1.65 −284.48± 1.47 −253.13 ± 1.34 −277.01 ± 1.83$\left\langle {E\frac{vdW}{enz}} \right\rangle$ −329.18 ± 0.47 −367.88 ±0.48 −321.53 ± 0.74 −356.63 ± 0.18$\left\langle {E\frac{vdW}{{enz} + {sub}}} \right\rangle$  −42.25 ± 1.15 −47.28 ± 1.82  −41.37 ± 1.18  −46.71 ± 1.09 * The binding free energy(ΔG)) calculation was based on the molecular dynamics simulation;${\Delta\; G} = {{0.2\left( {\left\langle {E\frac{vdW}{{enz} - {sub}}} \right\rangle - \left\langle {E\frac{vdW}{enz}} \right\rangle} \right)} - {0.5\left( {\left\langle {E\frac{Elec}{{enz} - {sub}}} \right\rangle - \left\langle {E\frac{Elec}{enz}} \right\rangle} \right)}}$wherein < > denotes averages of the van der Waals (wdW) andelectrostatic (Elec) interactions between the substrate (sub) such asUDP-Glc and its surrounding residues in enzymes (enz) such as KanF andNemD. 0.2 and 0.5 are scaling factor for wdW and Elec, respectively.

The inventors have discovered that the substrate-flexibleglycosyltransferase KanF synthesizes compounds 9 and 2 by taking bothUDP-Glc and UDP-GlcNAc as cosubstrates for attachment to compound 1. Thediscovery of the structure of compound 9 indicated that KanF is aglucose transferase as well as an N-acetylglucosamine transferase. Theactivity of this glycosyltransferase has not been known in thebiosynthesis of aminoglycoside. Moreover, compound 9 is converted intocompound 10 in the same way as compound 3 is converted into compound 4by KanI-KacL. KanI-KacL also convert compounds 6 and 12 into compounds 7and 8, respectively. The second glycosyltransferase KanE also showsremarkable substrate flexibility toward the glycosyl-acceptor andproduces compounds 6, 7, 12, and 8, respectively by transferring UDP-Knsto compounds 3, 4, 9, and 10. KanE also takes UDP-Glc as theglycosyl-donor and transfers UDP-Glc to the pseudodisaccharide, therebyproducing compounds 5, 13, 11, and 14 (FIGS. 2 and 3).

The method of directly producing AHBA-binding kanamycin using therecombinant strain of the present invention is first discussed as thedirect fermentative production of the semi-synthetic aminoglycoside.

Moreover, compound 17 (1-N-AHBA-kanamycin X) as a new aminoglycosidecompound produced by the present invention has strong antibacterialactivity against all kanamycin and amikacin-resistant test strains. Theunique structural difference between compound 15 and 17 is a functionalgroup attached at C-6′ position (FIG. 5C), which indicates that compound17 is active against bacteria having resistance to compound 15 byremoving the 6-amino group which acts as a target of aminoglycoside6-acetyltransferase. Another advantage of compound 17 is the reducedtoxicity due to the reduction in the number of amino groups.

Furthermore, the in vivo production of compound 16 by a modified pathwayis more economical than the conventional method using hydrolysis of6″-O-carbamoyltobramycin which occupies 9% of the total nebramycinfactors produced by S. tenebrarius.

MODE FOR THE INVENTION

Hereinafter, the present invention will be described by way of examplesin detail. However, the following examples are provided to facilitatethe understanding of the present invention, and the present invention isnot limited to or by the following examples.

Example 1 Preparation of Materials

Standard kanamycin, kanamycin B, neomycin, paromomycin, tobramycin, andamikacin were purchased from Sigma (USA). 2-mercaptoethanol,phenylmethylsulfonyl fluoride (PMSF), phenol/chloroform/isoamyl alcohol(25:24:1), uridine 5′-diphospho-D-glucose (UDP-Glc), and glass beads(150 to 212 μm) were also purchased from Sigma. Heptafluorobutyric acid(HFBA) was obtained from Fluka, and HPLC-grade acetonitrile, methanol,and water were obtained from J. T. Baker. 2-deoxystreptamine (2-DOS,compound 1) and UDP-2-N-acetyl-D-glucosamine (UDP-GlcNAc) were purchasedfrom GeneChem (Republic of Korea). Cation solid-phase exchanger (OASISMXC SPE, 3 mL/60 mg) and vacuum manifold were purchased from Waters. Theculture medium components, soybean meal, yeast extract, and malt extractwere acquired from BD Science (USA).

Paromamine and neamine were prepared from paromomycin and neomycin,respectively, by methanolysis. UDP-2-D-glucosamine (UDP-GlcN) wasprepared by enzymatic reaction of UDP-D-glucose pyrophosphorylase withglucosamine-1-phosphate and uridine 5′-triphosphate (UTP). Escherichiacoli DH10B and plasmid Litmus 28 (New England Biolabs) were used forroutine subcloning. High-copy number E. coli-Streptomyces shuttle vectorpSE34 containing the strong ermE* promoter plus a thiostreptonresistance marker was used as an expression plasmid.

Example 2 Culture of Strains

The recombinant strains of S. venezuelae were grown in liquid R2YE at30° C. for preparation of protoplasts, which were regenerated on R2YEagar medium supplemented with thiostrepton (30 μg/ml). The E. colistrains used for subcloning were grown in LB medium supplemented withampicillin (50 μg/ml) to select for plasmids.

For production of kanamycin biosynthetic intermediates and theiranalogs, S. venezuelae strains expressing the biosynthetic candidategenes were cultivated at 30° C. for 4 days in one liter of baffledErlenmeyer flasks containing 300 mL of R2YE medium supplemented withthiostrepton (25 μg/ml). S. kanamyceticus ATCC 12853 was grown at 30° C.for 5 days in liquid ISP2 (0.4% yeast extract, 1.0% malt extract, and0.4% glucose), and S. tenebrarius ATCC 17920 was grown at 30° C. for 5days in fermentation medium (2.0% glucose, 2.0% soluble starch, 4.0%soybean meal, 0.5% yeast extract, 0.5% CaCO₃, and 0.4% MgSO₄7H₂O, pH7.0).

To check the antibacterial spectra of kanamycin biosyntheticintermediates and their analogs, a total six kinds of Gram-negativebacteria such as kanamycin-sensitive (Kan^(S)) E. coli ATCC 25922,kanamycin-resistant (Kan^(R)) Pseudomonas aeruginosa ATCC 27853, Kan^(S)E. coli CCARM (Culture Collection of Antimicrobial Resistant Microbes,Korea) 1A020, Kan^(R) E. coli CCARM 1A023, amikacin-sensitive (Amk^(S))P. aeruginosa CCARM 2206, and amikacin-resistance (Amk^(R)) P.aeruginosa CCARM 2178 were used.

Example 3 Cloning and Construction of Expression Plasmids andRecombinant S. Venezuelae Strains

An engineered strain of S. venezuelae, which is deficient inbiosynthesis of the endogenous deoxysugar thymidine 5′-diphospho(TDP)-D-desosamine, was used as a heterologous host. A gene replacementplasmid, pYJ188, was introduced into protoplasts of S. venezuelae YJ003mutant for deletion of the kanamycin modifying gene (aphII) by areplicative plasmid-mediated homologous recombination. Several doublecrossover mutants were identified on the basis of their phenotypes ofkanamycin sensitivity and their genotypes by Southern hybridization.

DNA fragments containing a variety of kanamycin biosynthetic genes wereamplified from pSKC2 by PCR with specific deoxyoligonucleotide primers(See table 1). The DNA fragments encoding NemD, GtmG, AprD3, AprD4,TobM1, and TobM2 were obtained by PCR-amplification using genomic DNA ofS. fradiae ATCC 10745, Micromonospora echinospora ATCC 15385, and S.tenebrarius ATCC 17920, respectively. DNA fragments containing btrI,btrJ, btrK, btrO, btrV, btrG, and btrH required forS-4-amino-2-hydroxybutyric acid (AHBA) biosynthesis and transfer werePCR-amplified using genomic DNA of Bacillus circulans NR3312 as atemplate. Each pair of primers contained several restriction sites tofacilitate subcloning of each DNA fragment.

PCR was performed using Pfu polymerase (Fermentas) under themanufacturer's recommended conditions. All PCR products were cloned intoLitmus 28 and sequenced to confirm their authenticity.

For expression of 2-DOS biosynthetic genes, PCR-amplified fragmentscontaining kanA-kanB and kanK were digested with BamHI/SpeI andXbaI/HindIII, respectively, and simultaneously ligated with pSE34 whichhad been cut with BamHI and HindIII, thus generating pDOS. Plasmid pDOSf(kanA-kanB-kanK-kanF-gtmF-gtmK-gtmL) was constructed to express kanFalong with 2-DOS biosynthetic genes by ligating the SpellHindIII-digested DNA fragment of kanF with the XbaI/HindIII fragments ofresistance gene set (gtmF-gtmK-gtmL) isolated from pYJ489, and insertinginto pDOS as an XbaI/HindIII fragment.

All subsequent cloning steps for construction of plasmids pPAR, pPARcd,pPARil, pKCXΔcΔd, pKCX, pKABΔcΔd, pKAB, pDOSn, pDOSt₁, pDOSg, pKCXΔfn,pKCXΔft₁, pKCXΔfg, and pKABΔfn were performed using the compatiblecohesive ends of SpeI and XbaI sites.

For the direct fermentative production of 1-N-AHBA kanamycins, plasmidLitmus 28 containing btrG-btrH was digested by SpeI/SnaBI and ligatedwith the DNA fragment btrI-btrJ that had been cut by XbaI/SnaBI. Theresulting plasmid carrying btrG-btrH-btrI-btrJ was subsequently ligatedwith the DNA fragment containing btrK-btrO-btrV in the same manner.Then, the plasmid containing btrG-btrH-btrI-btrJ-btrK-btrO-btrV wasdigested by XbaI/SpeI and transplanted into the XbaI site of pKCX andpKAB (See table 2) to generate pKCXb and pKABb, respectively. Thecorrect orientations of the DNA fragments containingbtrG-btrH-btrI-btrJ-btrK-btrO-btrV in the pKCXb and pKABb were confirmedby restriction fragment analyses.

Next, plasmid pKABΔfna and pKABΔfnΔet₂a were constructed in attempt toproduce 3′-deoxykanamycins in vivo. The plasmid Litmus 28 containingaprD3 was digested by SpeI/HindIII and combined with the XbaI/HindIIIDNA fragment containing aprD4. The plasmid containing aprD3-aprD4 geneset was ligated with the XbaI/HindIII DNA fragments containingnemD-kanE-kanC-kanD-kanI-kacA-kacL-gtmF-gtmK-gtmL. The resulting plasmidwas moved into the XbaI/HindIII sites of pSE34 containing kanA-kanB-kanK(pDOS) to generate pKAB fna. Plasmid pKABΔfnΔet₂a was equivalent topKABΔfna supplemented with tobM2 instead of kanE.

To prepare the cell-free extract of S. venezuelae for functionalassignment of each kanamycin biosynthetic gene products, DNA fragmentscontaining kanF, kanE, kanI-kacL, kanC-kanD, and aprD3-aprD4 weretransplanted into pSE34 as XbaI/HindIII fragments, thus generatingpKanF, pKanE, pKanI-KacL, pKanC-KanD, and pAprD3-AprD4, respectively.All resulting plasmids were then transformed into an engineered strainof S. venezuelae, thus yielding the corresponding recombinants.

Example 4 Isolation and Identification of Kanamycin BiosyntheticIntermediates and their Analogs

HPLC was performed with a Spherisorb S5 ODS2 (250×20 mm, Waters)semi-prep column on the products of engineered recombinant strains inExample 3. The products were eluted with the same mobile phase used inHPLC-ESI-MS/MS analysis at a flow rate of 12 mL/min over a period of 150min. The resulting eluent was fractionated into 3-mL portions that weremonitored by HPLC-ESI-MS/MS to detect and characterize the presence ofeach kanamycin-related biosynthetic intermediate and analog. Fractionscontaining products of interest were pooled and extracted using OASISMCX SPE followed by freeze-drying. ¹H, ¹³C, and 2D ¹H-¹H COSY NMRspectra were acquired using a Varian INOVA 500 spectrometer at 298 K.Chemical shifts were reported in ppm usingtrimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP) as an internalreference. The assignment of each compound was carried out by comparisonwith previously assigned ¹H and ¹³C NMR spectra, and NMR data processingwas done using MESTREC (Magnetic Resonance Companion) software.

Example 5 Analysis of Kanamycin Biosynthetic Intermediates and Analogs

Kanamycin biosynthetic intermediates and their analogs produced byrecombinant S. venezuelae strains expressing aminoglycoside biosyntheticgenes were extracted from the fermentation medium using the OASIS MCX(Waters) SPE cleanup procedure and analyzed by HPLC-ESI-MS/MS.

Analytical HPLC-ESI-MS/MS was performed on the analytes in an XTerra MSC₁₈ column (50×2.1 mm, 3.5 μm, Waters). The analytes were eluted withacetonitrile and 10 mM heptafluorobutyric acid (Fluka) for 45 minutes.Quantification of the analytes was conducted using MS/MS in the multiplereactions monitoring mode. This was done by selecting the two mass ionsset to detect a transition of the parent ion to the product ion specificto the selected analytes: compound 1, 163>84; compound 2, 366>163;compounds 3 and 10, 324>163; compound 4, 323>163; compounds 5, 12 and14, 486>163; compounds 6, 8 and 13, 485>163; compound 7, 484>163;compound 9, 325>163; compound 11, 487>163; compound 15, 586>264;compound 16, 468>163; compound 17, 587>264; compound 18, 307>163; andcompounds 19 and 20, 469>163. Three separate cultivations andextractions were performed.

Example 6 In Vitro Reactions to Analyze AprD3-AprD4 Activity

In vitro reactions to analyze the activity of AprD3-AprD4 pair on3′-deoxygenation in the pseudodi- or tri-saccharide were carried out bysupplementing 100 μM neamine or kanamycin B with the cell-free extractsfrom the recombinant host expressing aprD3-aprD4. After incubation at30° C. for 2 hours, the reaction was quenched. The resulting supernatantcontaining the product of interest was extracted using OASIS MCX SPE,and then subjected to HPLC-ESI-MS/MS analysis. Independent experimentswere carried out in duplicate, and the results are shown in FIG. 7.

Example 7 In Vitro Reactions to Analyze Glycosyltransfer Activity ofKanF

Cell-free extracts of S. venezuelae were prepared by glass-beadhomogenization. These cell-free extracts were suspended in a Tris buffersolution containing 100 mM TrisHCl (pH 7.6), 10 mM MgCl₂, 6 mM2-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma),and each protein concentration was calibrated. The glycosyltransferreaction by KanF was initiated by supplementing 100 μM 2-DOS togetherwith 200 M UDP-Glc (Sigma), UDP-GlcNAc (GeneChem), or UDP-GlcN ascosubstrates to the cell-free extracts of the recombinant hostexpressing only kanF. 100 μM 6′-hydroxy pseudodi- and tri-saccharides 3,6, 9, and 12 were mixed with the cell-free extracts of the recombinanthost expressing kanI-kacL to measure the activity of KanI-KacL forC-6′-amination. The resulting cell-free extracts were incubated at 30°C. for 2 hours before quenching with ice-cold phenol/chloroform/isoamylalcohol (25:24:1, Sigma) and then subjected to centrifugation at 18,000g for 5 minutes. The supernatant containing the product of interest wasextracted using OASIS MCX SPE, mixed with 100 μM water, and thensubjected to HPLC-ESI-MS/MS analysis.

Example 8 In Vitro Reactions to Analyze KanC-KanD Activity

In vitro reactions to analyze the activity of KanC-KanD were carried outusing cell-free extracts of two S. venezuelae strains expressing kanE orkanC-kanD. 200 μM UDP-Glc and 100 μM compound 3 or 4 were mixed with thecell-free extracts of the strains expressing kanE (or kanC-kanD) andincubated under the same conditions as Example 7 before quenching thereactions. After the resulting supernatant containing the product ofinterest was incubated together with the cell-free extracts of thestrains expressing kanC-kanD (or kanE) at 30° C. for 2 hours, thereaction was quenched. The resulting supernatant was extracted in theabove manner and then subjected to HPLC-ESI-MS/MS analysis.

Example 9 In Vitro Reactions Using Cell-Free Extracts from Wild-TypeStrains of S. kanamyceticus and S. tenebrarius

In vitro reactions to determine the glycosyltransfer activities of KanEfrom S. kanamyceticus and TobM2 from S. tenebrarius on nebramine, wereperformed by supplementing 100 μM nebramine with the cell-free extractobtained from S. kanamyceticus (SK CFE) and TobM2 from S. tenebrarius(ST CFE). To minimize the presence of the kanamycin- ortobramycin-related congeners in the cell-free extracts of both wild-typestrains, which might interfere with reactions, the cell-free extractsderived from glass-bead homogenization were further treated using OASISMCX SPE pass-through as follows. At first, the cell-free extractsobtained from both wild-type strains were loaded onto each SPEcartridge, which was conditioned with methanol and water. Next, thepass-through from the cartridge was pooled, and the same step wasrepeated again. The above-mentioned cleanup procedures were all done inice-cold conditions of about 4° C. The resulting cell-free extracts wereincubated with compound 18 at 30° C. for 2 hours, and then subjected tothe OASIS MCX SPE cleanup and HPLC-ESI-MS/MS analysis. Independentexperiments were carried out in duplicate, and the results are shown inFIG. 8.

Reactions to determine the conversion of compound 6, 7, or 12 intocompound 8 were performed by supplementing 100 μM of compound 6, 7, or12 with the cell-free extracts obtained from S. kanamyceticus. Thesecell-free extracts derived from glass-bead homogenization were furtherpurified using OASIS MCX SPE pass-through as described above. Afterincubation at 30° C. for 2 hours, the reaction was quenched. Theresulting supernatant containing the product of interest was extractedusing OASIS MCX SPE, and then subjected to HPLC-ESIMS/MS analysis.Independent experiments were carried out in duplicate, and the resultsare shown in FIG. 9.

Example 10 Measurement of MIC of Kanamycin Biosynthetic Intermediatesand Analogs

Minimum inhibitory concentrations (MICs) of various kanamycin-relatedpseudodi- and tri-saccharides and AHBA conjugated kanamycin analogs(except for compounds 16, 18, 19, and 20) were measured by brothmicrodilution according to the Clinical and Laboratory StandardInstitute (CLSI, formerly NCCLS). As described in Example 2,gram-negative E. coli strains, P. aeruginosa strains, and clinicallyisolated strains were incubated in Mueller-Hinton broth (BD Science) at30° C. Then, the aminoglycoside produced in the present invention wasdiluted 2-fold serially to give final concentration between 0.25 to 128μg/ml. An aliquot of water was used as a negative control group. Thegrowth of the strains was observed using the Labsystems Bioscreen C, andthe minimum concentrations of the aminoglycoside diluted in the brothmedium to inhibit the growth of the bacterial strains were measured.

Example 11 3-Dimensional (3D) Implementation for GlycosyltransferasesSuch as KanF and NemD

Homology modeling of a couple of glycosyltransferases such as KanF andNemD was performed with MODELLER and optimized by FoldX using the atomiccoordinates of MshA (PDB code: 3C48) as a template. The 3D structuremodels were assessed by VADAR program. Molecular docking was employed todetermine the binding conformation of the glycosyl donors (UDP-Glc andUDP-GlcNAc) and acceptor (2-DOS), and energy-minimized andstructure-optimized with Gaussian03 using HF/6-31G(d) basis set formolecular dynamics simulation.

The docking of the glycosyl donors and 2-DOS to glycosyltransferases wasinitially conducted according to the predicted topological binding sitesby several algorithms. The automated docking was carried out by CDOCKERprogram (Accelrys, Inc.) based on the MMFF force field and AutoDock 4.0program suite. The active site was defined with 6 Å radius sphere fromthe putative catalytic center of KanF and NemD. Each complex model wassolvated with TIP3 water molecules in a cube box and ensured the wholesurface of KanF and NemD protein with their glycosyl donors/2-DOS to becovered by a water layer with a thickness more than 12 Å The energyminimization for each complex was performed using the steepest descentalgorithm, followed by the conjugate gradient in the CHARMM. Then, a500-ps position restrained molecular dynamics was performed with theproteins and glycosyl donors/2-DOS using CHARMM package. Finally, a 3nanoseconds molecular dynamics was started by taking initial velocitiesfrom a Maxwellian distribution at 300 K. Solvent and glycosyldonors/2-DOS were independently, weakly coupled to a temperature bathwith a relaxation time of 0.1-ps. The system was also isotropically,weakly coupled to a pressure bath at 1.0 atm with a relaxation time of0.5 picoseconds and an isothermal compressibility of 0.5 10⁻³/bar.Long-range electrostatics was calculated by the particle-mesh Ewaldmethod. Short-range van der Waals and coulombic interactions were cutoff at 0.1 Å. All bond lengths were constrained using the SHAKEalgorithm, and the time step was set to 0.002 picoseconds. The bindingfree energies between the glycosyl donors and KanF (or NemD) werecalculated using the linear interaction energy method using defaultparameters.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

[Accession Nos.]

KABΔfn strain: KCTC11725BP

KABΔfnΔet₂a strain: KCTC11726BP

KCXb strain: KCTC11727BP

KABb strain: KCTC11728BP

INDUSTRIAL APPLICABILITY

According to the present invention, a kanamycin-producing Streptomycesspecies bacterium, a method of effectively producing kanamycin, and anew kanamycin compound produced by the same are provided.

The invention claimed is:
 1. An isolated compound of formula 1 or a pharmaceutically acceptable salt thereof:


2. A pharmaceutical composition comprising the isolated compound of formula 1 or a pharmaceutically acceptable salt thereof, according to claim 1, and a pharmaceutically acceptable excipient.
 3. A method of treating a bacterial infection or a viral infection in a patient comprising administrating to the patient a composition comprising the isolated compound of formula 1 or a pharmaceutically acceptable salt thereof, according to claim
 1. 4. The method of claim 3, wherein the bacterial infection is a Gram-negative bacterial infection.
 5. The method of claim 3, wherein the bacterial infection is a Pseudomonas aeruginosa infection or an Escherichia coli infection.
 6. The method of claim 5, wherein the P. aeruginosa is amikacin-resistant P. aeruginosa. 